1 Project Summary/Abstract The availability of simplified and better-tolerated antiretroviral therapy (ART) drug combinations has decreased disease burden in infected individuals and reduced transmission to their contacts. However, treatment for HIV infections requires life-long antiviral therapy since current drug treatments do not lead to ?cures?. The single most significant obstacle to total HIV clearance is that of viral latency since the presence of a silent HIV genome in human cells is not recognized as infected by the immune system or by any available antiviral drugs. Thus, the largest challenge to the HIV/AIDS basic sciences research community today is to determine targets and strategies to fully eradicate virus from the body by eliminating this reservoir pool of latently infected cells. One strategy to eliminate latently infected cells involves activating the latent provirus with drugs called latency reversal agents (LRAs) in the presence of suppressive ART, and then using immunological methods to kill the cells newly-producing virus. However, we lack a comprehensive and complete understanding of how HIV latency is established and maintained, and how reactivation occurs across cell types. Importantly, no single genetic or chemical intervention has been able to induce activation of all latent proviruses (or even the majority of them). This proposal describes a novel and powerful approach to understanding HIV latency based on an innovative genetic screening method that we have developed called HIV-CRISPR that uses the packaging of CRISPR guides into budding HIV to serve as a readout for the effects of host genes on HIV. This technique is both high-throughput and comprehensive and our plan is to adapt to models of HIV latency. Because the screening technology is versatile and can be done in many iterations, we will be able to adopt a strategy based on combining genetic screens with low doses candidate latency reversal agents to identify synergism between different pathways of HIV latency maintenance in order to find targets that more broadly activate HIV from latency while at the same time providing a greater degree of specificity for the HIV LTR. Moreover, we will develop methods this technique to primary cell models of HIV latency to better mimic how to reverse latency in vivo. Finally, will also apply this screen to understand how drugs of abuse influence HIV latency pathways.